tbuckley/cabot-search huggingface dataset:
Abstract P3130: Preventing Site-specific Calpain Cleavage Of Junctophilin-2 Protects Against Stress-induced E-c Coupling Dysfunction And Heart Failure Development In Mice
Authors: Jinxi Wang
Journal: Circulation Research (2023)

In heart failure (HF), excitation-contraction (E-C) coupling processes become disrupted, resulting in abnormal Ca 2+ homeostasis that triggers cardiac dysfunction. Junctophilin-2 (JP2) is an essential E-C coupling constituent for maintaining cardiomyocyte integrity and cardiac function both at baseline and in response to stress. We previously showed that calpain-mediated proteolytic cleavage of JP2 at arginine 565 / threonine 566 (R 565 /T 566 ) is key to its downregulation in the diseased heart. However, it remains unclear whether preventing site-specific proteolysis of JP2 can prevent cardiac remodeling and HF progression following stress. To investigate this, calpain-resistant mutant JP2 knockin mice (JP2 CR ) were generated by deleting residues 563-568 spanning the primary calpain cleavage site. Studies in cultured cardiomyocytes treated with isoproterenol (ISO) showed that JP2 CR is resistant to stress-induced loss of JP2 integrity and T-tubule disorganization compared to wild-type (WT) cardiomyocytes. Further, in vivo studies demonstrate JP2 CR mice are more resistant to pressure overload stress having significantly less abnormal Ca 2+ homeostasis, cardiac dysfunction, hypertrophy, lung edema and fibrosis relative to WT mice. Western blot analysis revealed JP2 CR hearts have preserved expression levels of several key E-C coupling proteins, including RyR2, SERCA2a and Ca V 1.2. RNA-Sequencing analysis further revealed attenuated pressure overload induced hypertrophic and heart failure-related transcriptional reprogramming in JP2 CR hearts. These findings indicate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain-cleavage of JP2 offers cardiac protection against stress-induced hypertrophy and heart failure. Our data indicate that preventing JP2 cleavage by gene therapy approaches may hold great therapeutic potential for treating heart failure.
Abstract P2035: Rad Reduction Improves Heart Failure
Authors: Jonathan Satin, Garrett Elmore, Brooke Ahern, Bryana Levitan, Jessica Miller, Riham Abouleisa, Robert N Correll, Alec Dupont, Yuan Wen, Andrea Sebastian, Erhe Gao, Tamer Mohamed, Douglas A Andres
Journal: Circulation Research (2023)

Background: Heart failure with reduced ejection fraction (HFrEF) entails depressed systolic function. Targeting calcium handling provides a direct, restorative treatment for the loss of systolic function. We recently showed that induced, cardiomyocyte-restricted Rad knockout mice (cRadKO) show increased trigger Ca 2+ with improved contractility and no signs of pathological remodeling. Hypothesis: In the present study we tested the hypothesis that cRadKO provides inotropic support to hearts subjected to chronic pressure overload. Results: We first show that Rad-reduction in human myocardial slices provides an inotropic boost. We then performed transverse aortic constriction (TAC) in mice. cRadKO imposed either before TAC or 1-month after TAC promoted survival and attenuated cardiac remodeling. cRadKO induced after TAC reflects an interventional proof-of-principle that Rad reduction can attenuate heart failure progression. The intervention timeline group was further probed using bulk RNA sequencing. cRadKO attenuated key fetal gene program transcripts including NPPA, NPPB, and MYH7. RCAN1 levels were decreased with cRadKO consistent with reduced calcineurin-NFAT signaling, and BIN1 levels were restored in cRadKO, suggesting improved cardiac remodeling despite ongoing pressure overload. Conclusions: The prevention (cRadKO before TAC) and intervention (cRadKO after TAC) studies demonstrate that reduced Rad levels can provide an inotropic boost in HFrEF and stimulate transcriptional effects consistent with reparative remodeling.
Abstract P2163: The Genetic And Pharmacological Inhibition Of Classic Transient Receptor Potential Channels 3 And 6 (trpc3/c6) Improve Pulmonary Arterial Hypertension.
Authors: Kenji Moriuchi, Yasuaki Nakagawa, Hideyuki Kinoshita, Hideaki Inazumi, Hiromu Yanagisawa, Takahiko Kanamori, Toshio Nishikimi, Miku Oya, Motohiro Nishida, Yasuo Mori, Ryu Nagata, Kazufumi Nakamura, Koichiro Kuwahara, Kazuwa Nakao, Takeshi Kimura
Journal: Circulation Research (2023)

Background: Pulmonary artery hypertension (PAH) is a progressive and fatal disease. Currently, there are only three types of PAH drugs, targeting the endothelin pathway, NO pathway, and prostacyclin pathway, and all these drugs are pulmonary vasodilators. However, the pathogenesis of PAH develops through various mechanisms, including vascular endothelial dysfunction, pulmonary artery smooth muscle cell (PASMC) proliferation, fibrosis and inflammation as well as vasoconstriction. Recently, calcium signaling has been reported to be involved in these multiple mechanisms in PAH. Classic transient receptor potential channel 3 and 6 (TRPC3/C6), known as receptor-operated calcium channels, have been reported to be involved in the pathogenesis of PAH. Methods and Results: Initially, we evaluated TRPC3 knockout mice (TRPC3KO), TRPC6 knockout mice (TRPC6KO), and TRPC3/C6 double-knockout mice (dKO) in the PAH models, to evaluate the effects of genetic inhibition of TRPC3/C6. All of TRPC3KO, TRPC6KO, and dKO have weakened PAH phenotypes such as the systolic pulmonary artery pressure, the right ventricular hypertrophy, and the tunica media thickening of the pulmonary artery in monocrotaline pyrrole-induced PAH models. On the other hand, in the hypoxia-induced PAH model, TRPC6KO and dKO ameliorated these PAH phenotypes, but TRPC3KO didn’t. Next, we evaluated the effect of pharmacological inhibition of TRPC3/C6 in PAH model rats. We have developed a novel selective TRPC3/C6 inhibitor, L862. This compound showed excellent pharmacokinetic and safety properties. L862 ameliorated monocrotaline-induced and hypoxia-induced PAH in rat models on prevention protocol. Moreover, L862 significantly suppressed PDGF-BB-induced proliferation of human PASMC derived from idiopathic PAH patients in a dose-dependent manner. L862 also improved PAH in Sugen/hypoxia rat model on the therapeutic protocol. Conclusions: Both genetic and pharmacological inhibition of TRPC3/C6 ameliorated PAH in several PAH models. These results suggested that TRPC3/C6 had essential roles in the pathogenesis of PAH and L862 had potential clinical application.
Abstract P1064: Gene Editing Technologies To Understand Knockout And Over Expression Of Circadian Rhythm Regulatory Genes In Human Derived Spheroid Model.
Authors: Saidulu Mattapally, Wesley C Labarge, Gregory Walcott, Chandrakala Mattapally, Jianyi J Zhang
Journal: Circulation Research (2023)

Introduction: Human induced pluripotent stem cells (hiPSCs) have the ability to produce a huge variety of terminally differentiated cell types that can be used for tissue engineering, drug research, and cell therapy. Using CRISPR/Cas9 gene-editing technologies, hiPSCs with knockout mutations for Per1, Per2, CIITA, B2M, N-Cadherin, Connexin43 and over expression of Connexin43 were created. Cardiomyocytes (CM) were created from hiPSCs, and spheroid culture was carried out as previously described. The molecular basis of circadian regulation of human induced pluripotent stem cells (hiPSCs) is still under investigation. Methods: From hiPSC CMs, we created and described a 3D spheroid culture. We assessed the effectiveness of spheroid transplantation for cardiac healing in immunecompromised mice and pigs. We investigated border zone macrophage markers, border zone vascular density, and cell proliferation in myocardial infarction after spheroid transplantation. We used pedigree analysis and 20 years of family health diagnostics data to analyze the clinical consequences of type 2 diabetes medications among metformin combo patients in big extended family (N=30).. Results: TNF-Alpha, IL1-beta, NOS2, and IL-6 relative expression were shown to be lower in the KO group than in the WT group. Significant engraftment of hiPSC-derived cardiac spheroids was seen up to 4 weeks after transplantation, according to histological examination. Arrhythmogenicity is significantly altered by the spheroid therapy. Ventricular tachycardia may be related to macrophage immunological regulation. To further understand, we looked at the effects of a medication combination of dapagliflozin and metformin in a large extended family, where 5 out of 30 members had type 2 diabetes. One patient experienced mildly tachycardic heart issues over the course of a 20-year medication treatment. Conclusion: Our study established the promising therapeutic potential of spheroids model from hiPSC differentiated CMs to understand Circadian rhythm gene regulation.
Abstract P2036: Induced Pluripotent Stem Cell- Derived Cardiomyocytes Provide New Insights Of The Pathogenic Consequences Of The Novel Frameshift Variant <i>(c.5769delG)</i> In <i>MYH7</i> Gene
Authors: Sara Elshorbagy, Ayman M Ibrahim, Fiesal Mohamed, Jerome Fourre, Barrett Downing, Manar Osman, Hasnaa A. Elfawy, Mariam Fathy, Mona Allouba, Yasmine Aguib, Cesare M Terracciano, Magdi Yacoub
Journal: Circulation Research (2023)

Background: the genetic basis of hypertrophic cardiomyopathy is pleiotropic; A novel disease causing frameshift variant (c.5769delG, MYH7 ) in the beta-myosin heavy chain gene has been recently described. However, the mechanisms involved have not been adequately defined. We have used iPSC-CMs to investigate the underlying mechanisms. Methods: We have generated patient-specific iPSCs from clinically and genetically defined HCM patients carrying the pathogenic variant (c.5769delG) (n=3) and compared them to iPSCs from healthy controls (n=3). Optical mapping of calcium transients and cell action potential were performed under different stimulation conditions. iPSC-CMs contractility was recorded via widefield microscopy. Gene and protein expression analyses were performed using RT-PCR, immunoblotting and immunocytochemistry. RNA sequencing data from patients’ myocardial tissues was analyzed to validate gene expression data from iPSC-CMs. Results: HCM iPSCs-CMs showed a significant decrease in calcium release duration (i.e., time to peak) with decrease in calcium transient amplitude, compared to controls. This was associated with an accelerated contraction and a decreased contraction amplitude. Nonetheless, action potential analysis showed no significant changes. Transcriptome analysis of iPSC-CMs and patients’ myocardial tissues showed a significant down regulation of SERCA/ATP2A2 , MYH6 and KCNIP2 . Conclusion: iPSC-CMs provided new insights of the pathogenic consequences of the novel variant in MYH7 gene on calcium handling and contractility. Key words: Hypertrophic Cardiomyopathy, Disease model, MYH7 , iPSC-CMs, Calcium Handling, Contractility.
Abstract P2153: A Long Noncoding RNA Plentiful In Cardiosphere-derived Cell Extracellular Vesicles (CDC-EVs), BCYRN1, And Its Short Synthetic Derivatives Enhance Regulatory T Cell (Treg) Proliferation, Migration, And Activation
Authors: ke liao, Zahra Mohammadigoldar, Akbarshakh Akhmerov, Ahmed G Ibrahim, Eduardo Marban
Journal: Circulation Research (2023)

Background: By modulating inflammation, Treg cells influence outcomes in myocardial infarction (MI). Here, we test the hypothesis that BCYRN1 (Brain Cytoplasmic RNA 1) and BCYRN1-derived binding small sequences (BDSSs) are cardioprotective by activating Tregs. Methods: RNA sequencing identified BCYRN1 as plentiful in cardiosphere-derived cell extracellular vesicles (CDC-EVs), validated by qPCR. RNA pulldown and luciferase assays probed BCYRN1's potential function as a miRNA sponge. We assessed BCYRN1 and BDSS effects on Treg proliferation (Cell Counting Kit-8), migration (trans-well migration), and IL-10 (qPCR and ELISA). In vivo , MI was induced in C57BL/6 mouse by left coronary artery ligation for 45 minutes followed by 15 minutes reperfusion. Afterwards, mice received (via tail vein) CDC-EV-BCYRN1 (CDC-EVs overexpressing BCYRN1), BDSSs, mut-BDSSs (scrambled inactive versions of BDSS) or vehicle (PBS). MI size (by TTC staining) and infiltrating Treg (by flow cytometry) were assessed 72 hrs post-MI. Findings: BCYRN1 was found to contain putative binding sites for miR-138, miR-150 and miR-98; these miRs target ATG7, CCR6 and IL-10 mRNAs, respectively. BCYRN1 serves as a miRNA sponge, leading to Treg cell ATG7/autophagy dependent proliferation (3.0± 0.1 fold increase, N=5), CCR-6 dependent migration (1.8 ± 0.1 fold increase, N=5) and increased IL-10 production (fold increase, qPCR: 2.6 ± 0.4, N=6 and ELISA: 6.1 ± 0.8, N=5). Lipid nanoparticles containing a cocktail of 3BDSSs likewise induce Treg cell proliferation (1.9± 0.6 fold increase, N=6), migration (1.3± 0.2 fold increase, N=6), and IL-10 induction (6.8± 0.5 fold increase, N=6). In vivo (N=5/group), CDC-EV-BCYRN1 (but not vehicle) and BDSS cocktail (but not mut-BDSS) induced Treg infiltration, proliferation and IL10 production in the heart with a concomitant decrease of infarct size. Conclusions: A single LncRNA (BCYRN1) and shorter synthetic derivatives mimic the effects of CDC-EV on Treg in vitro and in vivo . Our study provides a novel approach, targeting Treg, to treat MI. This approach could be useful for other diseases where increased Treg activity is a therapeutic goal (e.g. autoimmune disorders).
Abstract P2154: A Novel Allosteric Modulator Of The β <sub>1</sub> AR Identified By DNA-encoded Small Molecule Library Screening Demonstrates Unique Pharmacology And Function
Authors: Alyssa Grogan, Seungkirl Ahn, David Israel, Alex Shaginian, Qiuxia Chen, Jian Liu, Max Wilkey, Jialu Wang, Alem Kahsai, Robert J Lefkowitz, Howard A Rockman
Journal: Circulation Research (2023)

While traditional β-blockers (i.e. competitive orthosteric antagonists of β-adrenergic receptors; βARs) are widely used as cardiovascular therapeutics, adverse effects such as fatigue and the nonselective inhibition of multiple receptor subtypes often limit maximal effectiveness. An emerging approach to enhance therapeutic targeting is to identify allosteric modulators that act cooperatively with orthosteric ligands. In contrast to orthosteric ligands which bind the endogenous ligand binding site, allosteric modulators bind to regions that are topographically distinct from the orthosteric pocket and can enhance (positive allosteric modulator; PAM) or reduce (negative allosteric modulator; NAM) the activities of orthosteric agonists/antagonists. Since allosteric regions exhibit greater sequence and structural diversity among receptor subtypes relative to the more highly conserved orthosteric pocket, allosteric modulators are more likely to be subtype specific and/or generate less adverse effects. We therefore embarked on a DNA-encoded small molecule library screen to identify novel allosteric modulators of the β 1 AR. Following multiple rounds of affinity selection using purified, functional, β 1 ARs reconstituted in lipid nanodiscs and HitGen’s OpenDEL® small molecule library containing more than 1 billion unique compounds, we identified Compound 11 (C11) as an allosteric modulator with unique pharmacological properties. Notably, C11 binds to the β 1 AR with micromolar affinity and enhances the binding affinity of orthosteric agonists and certain antagonists to the β 1 AR. In contrast to its positive cooperative effect on ligand binding, cell signaling assays showed C11 potently inhibits G protein and β-arrestin signaling downstream of the β 1 AR. Importantly, C11 showed high β 1 AR specificity with no effect on β 2 AR or AT 1 R signaling. These results suggest that C11 is a β 1 AR-specific PAM in terms of ligand binding but a NAM in terms of agonist efficacy, belonging to a largely under-characterized class of allosteric modulators termed PAM-antagonists. With an extremely unique pharmacological profile, C11 is a promising potential therapeutic and experiments evaluating its ability to modulate β 1 AR signaling in vivo are ongoing.
Abstract P3122: <i>Rap1gap2</i> Fine-mapping For Causal Variants Of Prevalent Ischemic Heart Disease Among Diverse Postmenopausal Women From The Women’s Health Initiative
Authors: Jennifer R Dungan, Huaizhen Qin, Ayumi Franco-Sazaqui, Matthew Eynon, Simon G Gregory, Rhonda M Cooper-Dehoff, Carl J Pepine
Journal: Circulation Research (2023)

Background: RAP1GAP2 is a GTP-ase activating protein that regulates secretion of dense granules from platelets at sites of endothelial damage, eliciting maximal platelet aggregation, a hallmark of ischemic events. Our published sex-stratified GWAS significantly associated RAP1GAP2 (Chr 17p13.3) variants with ischemic heart disease (IHD) case status and increased mortality risk among women but not men. We hypothesize that RAP1GAP2 genetic variation may demonstrate causal effects for the increased risk of IHD case status among postmenopausal women. Purpose: To fine-map RAP1GAP2 to identify causal markers for risk of IHD case status among racially and ethnically diverse postmenopausal women. Methods: We performed secondary analysis of 8,699 postmenopausal women from the Women’s Health Initiative (Observational cohort) who have publicly available genome-wide and densely imputed genotype data via NIH/dbGap (Affymetrix Affy 6.0). IHD case status was defined as prevalence of myocardial infarction, stroke, angina, congestive heart failure, peripheral arterial disease, or atherosclerotic interventional procedures at or prior to baseline; non-IHD controls were absent of all IHD criteria. PCA and Efficient Local Ancestry Inference (ELAI) were used to infer global and SNP-wise local ancestries. For each SNP, we performed SNP-wise logistic regression modeling, calibrating for the confounding effects of local and global ancestries and controlling for major cardiac risk factors, hormone replacement and cardiac medication exposure, IHD type (obstructive, nonobstructive), eGFR, education, income, and occupation. Results: Mean age 64.08 (±7.74) for a sample of 3,125 (36%) non-Hispanic Whites, 3,841 (44%) African Americans and 1,733 (20%) Hispanic Americans. We observed 1,932 IHD cases and 6,767 non-IHD controls. Novel PCA and ELAI uncovered strong spatial global population structure, varying ancestries, and ancestry-genotype correlations across genomic regions. Fine mapping analyses are underway and results will be available at time of presentation. Conclusions: Identifying sex- and ancestry-related causal genetic factors can serve as future targets for precision prevention of IHD among postmenopausal women.
Abstract P3017: TNF-α-mediated Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 Expression In Endothelial Cells
Authors: Florian Kleefeldt, Lisa Götz, Uwe Rueckschloss, Süleyman Ergün
Journal: Circulation Research (2023)

The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a central regulator of vascular physiology. Previously we have shown that progressive age leads to a mutual upregulation of vascular CEACAM1 and TNF-α expression that maintains a chronic pro-inflammatory milieu in the vasculature. This age-dependent CEACAM1 upregulation is crucial to vascular aging processes, e.g. oxidative stress and arterial collagen deposition. In the current study, we analyzed the signaling pathways that are involved in the TNF-α-mediated CEACAM1 expression. Using endothelial cell cultures, we conducted pharmacological as well as biochemical experiments. We found that the upregulation of endothelial CEACAM1 expression in response to TNF-α stimulation shows a biphasic pattern. Inhibitor studies point to an early response that is mediated by activation of NFκB, whereas at later time-points the persistent upregulation might depend on Catenin-driven expression. Our data support a novel mechanism of CEACAM1 expression regulation. Especially in inflammatory settings with enhanced TNF-α signaling, i.e. atherosclerosis or cancer that regulation might significantly be involved in the pathology.
Abstract P3019: During Salt-sensitive Hypertension, CD8+ T Cells Increase Renal Sodium Transporters, NKCC2 And ENaC
Authors: Katherine Deck, Yunping Guo, Lance Benson, Yunmeng Liu, Christoph Mora, Shengyu Mu
Journal: Circulation Research (2023)

Hypertension originates in part from impaired salt handling by the kidneys. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts) adoptively transferred from salt-sensitive hypertensive mice to normotensive mice stimulate Sodium Chloride Cotransporter (NCC) increasing sodium retention. However, whether other sodium transporters within the nephron are affected by CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize CD8Ts will also have an impact on other sodium channels. Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. In vitro, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously. In vivo, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. As for in vitro, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological or with additional corticosteroid levels. Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention and any effect of CD8Ts in increasing ENaC may be masked in physiologic conditions.